1. Field of the Invention
The present invention relates to a method for detecting human papillomaviruses (HPV) in biological material and to nucleotide sequences which are used in the method.
This method comprises the steps of:.                a) extracting/isolating nucleic acids from biological material, and        b) detecting HPV-specific DNA in the isolated nucleic acids.        
2. Related Prior Art
Various publications have disclosed a method of this nature.
Infections with HPV are connected with a large number of diseases of the skin and the mucous membrane. These diseases are usually benign tumors which appear as condylomas, warts or papillomas which as a rule heal spontaneously after a period lasting from months to years. With the aid of molecular biology and genetic manipulation it has also been possible to demonstrate the connection which exists between a variety of cancers and infections with papillomavirus. These tumors are, in particular, carcinomas of the anogenital region, i.e. particularly carcinomas of the uterine cervix but also skin carcinomas in the case of immunosuppressed patients or patients suffering from the rare disease of epidermodysplasia verruciformis (EV). It has by now become generally accepted that HPV is causally involved in the cancers in the region of the anogenital tract. All over the world, HPV DNA is detected in virtually all uterine cervical carcinomas and precursor lesions.
More than 100 HPV genotypes have by now been identified, with the clinical syndromes which can be caused by the infection frequently being characteristic of a particular HPV type. Vulgar warts of the limbs and plantar warts are caused, in particular, by HPV types 1, 2, 4, 10, 27, 28, 57 and 65. In accordance with their oncogenic potential, the EPVs which are connected with cancers in the anogenital region are subdivided into “low-risk” (e.g. 6, 11 and 40) and “high-risk” HPV types (e.g. 16, 18, 45 and 56). Low-risk HPV types principally cause benign tumors of the external genital region (condylomas) and low-grade intraepithelial neoplasias and are only rarely associated with carcinomas. On the other hand, the presence of high-risk viruses in epithelial cells of the anogenital tract constitutes a high risk factor for the development of a carcinoma. HPV types 5 and 8 are regarded as being to a large extent certain to be involved in the development of skin carcinomas in EV patients.
Cancer of the uterine cervix and certain forms of skin cancer are avoidable diseases if early diagnosis and treatment are ensured. A method which reliably detects the presence of an HPV infection, which encompasses all the HPV types and which, in addition to this, enables the virus to be typed, is therefore a prerequisite for selective therapy.
Histological methods (cytology screening) have been used since the 1950s for detecting precancerous lesions and precociously invasive carcinomas. The use of these methods resulted in a decline in the incidents of cervical cancer. Despite this, the cytology is methodologically inadequate, thereby leading both to falsely negative (invasive carcinomas 15% to 55%, precociously invasive carcinomas 20% to 70%) and falsely positive results (5% to 15%, thereby causing unnecessary anxiety to the patients). These inadequacies are:                Subjective errors—the cytology is complex; it requires intensive visual examination of cells. This technique is not objective, nor is it geographically standardized.        Erroneous or faulty sample isolation can lead to falsely negative or unclear results.        Histological methods generally only record the existing state of a tissue, which means that these methods lack prognostic value.        
As a result of the detection methods having been improved, it has by now become possible to detect the presence of papilloma-virus directly in infected tissues:
The method termed HCM (hybrid capture microplate), which is a test which can be obtained commercially from Digene Corp., Gaithersburg, Md., USA, is a signal-amplifying hybridization method. This method makes it possible to qualitatively detect 18 HPV types in uterine cervix tissue samples. The hybridization probes which are used are HPV-specific RNA sequences which cover the entire viral genome.
Incubating these probes with denatured HPV DNA from the infected tissue results in the formation of RNA/DNA hybrids, which can be detected using a specific antibody. The enzyme alkaline phosphatase, which is conjugated to the antibody, is used to detect the virus by means of chemiluninescence, after an appropriate substrate has been added.
The HCM method makes it possible to differentiate between two HPV DNA groups: HPV types 6, 11, 42, 43 and 44, which have a low carcinogenic potential, and HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68, which have a medium to high carcinogenic potential.
It is not possible to use, this method to type the papillomaviruses precisely. A disadvantage of this test method is, furthermore, that the use of an RNA mixture frequently results in cross reactions taking place between the two HPV classes. This can lead to falsely negative or falsely positive results. Another disadvantage is that this method does not detect the HPV 5 and 8 papillomaviruses.
Surentheran et al., “Detection and typing of Human Papilloma Viruses in mucosal and cutaneous biopsis from immunosuppressed and immunocompetent patients and patients with Epidermodysplasia verruciformis: A unified diagnostic approach”, Journal of Clinical Pathology 1998; 51:606-610, describe a polymerase chain reaction (PCR)-based method for detecting HPV DNA in skin biopsies and mucous membrane biopsies from immunosuppressed patients, from immunocompetent patients and from EV patients. For this, the authors use a variety of PCR primer pairs which all lie within the open reading frame of the viral gene encoding the structural protein L1.
The papillomaviruses are typed by subsequently analyzing the sequence of the amplified gene fragment. Recent investigations have shown that the L1 gene is less conserved, over all the known HPV types, than was originally assumed. For this reason, this method only succeeds in detecting a limited number of HPV types (HPV 2-6, 8, 10, 11, 16, 31, 41 and 57). There has been no report on whether is it possible to use this method to detect the high-risk HPV types 18, 45 and 56.
Jacobs et al., “Group-Specific Differentiation between High- and Low-Risk Human Papillomavirus Genotypes by General Primer-Mediated PCR and Two Cocktails of Oligonucleotide Probes”, Journal of Clinical Microbiology 1995, 33, 901-905, also use a primer pair, designated GP5+/GP6+, which lies within the viral gene encoding the L1 structural protein. The region of the L1 gene which is amplified in the PCR using this primer pair is 150 base pairs (bp) in length. Following gel-electrophoretic fractionation, specific probes can be used to assign the amplificates to the high-risk HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 54, 56 and 58 and to the low-risk HPV types 6, 11, 34, 40, 42, 43 and 44. The specific hybridization probes are in each case 30 bases in length.
Tieben et al., “Detection of Epidermodysplasia verruciformis-like human papillomavirus types in malignant and premalignant skin lesions of renal transplant recipients”, British Journal of Dermatology 1994; 131, 226-230, describe a method for detecting HPV DNA in skin tumors of patients following a kidney transplant, which method is likewise PCR-based. The various PCR primer pairs which are used in this instance lie within the conserved, virus-specific E1 gene, which encodes an ATP-dependent DNA helicase. By means of using four different PCR primer pairs, the authors succeed in detecting the following HPV types: HPV 1-5, 6b, 7-9, 10a, 11, 12, 14a, 15, 16-22, 24, 25, 31, 33, 36-38, 46, 49, 50 and 65. However, the inventor of the present application has found that the primer pair CP4/CP5 is not suitable for amplification.
In this publication, typing is effected by sequencing the amplificate. In this connection, it is noteworthy that only three of the abovementioned primer pairs can be used if the PCR is to be followed by a typing of this nature. The abovementioned primer pair CP4/CP5 cannot be used. Using this method, Tieben et al. succeed, by typing, in specifically detecting the HPV types 1a, 2a, 3-5, 6b, 8, 10a, 11-13, 14a, 15-18, 20-22, 24, 25, 31, 33, 36, 39, 41, 42, 46, 47, 49, 50, 57, 58, 63 and 65.
The advantage as compared with the method described by Surentheran et al. is that, in this present method, a larger number of different HPV types can be identified with one primer pair. Despite this improvement in the specificity, it is not possible to use this method to type a large number of HPVs, in particular the high-risk papillomaviruses HPV 45 and 56 and also HPV types 35, 51 and 52, which have medium oncogenic potential.
However, as far as the inventor of the present application is aware, the primer pair CP4/CP5 is not used routinely in hospitals; on the contrary, hospitals are currently only using primer pairs which lie within the L1 gene, such as the primer pair GP5+/GP6+ described by Jacob et al., loc. cit.